Methods for treating cell proliferative disorders and viral infections

ABSTRACT

The present invention concerns methods for treating cell proliferative diseases, tumors associated with viral infections, and certain viral infections. The disclosed methods use compounds which inhibit heat shock protein 90 proteins. Such methods block Rb negative or deficient cells in the G2/M phase of the cell cycle and rapidly causes their destruction.

This application is a 371 national phase of PCT application Ser. No.PCT/US01/23640 filed Jul. 27, 2001, and claims the benefit of U.S.Provisional Applications Ser. No. 60/221,415 filed Jul. 28, 2000 and60/245,264 filed Nov. 2, 2000.

BACKGROUND OF THE INVENTION

The eukaryotic heat shock protein 90s (HSP90s) are ubiquitous chaperoneproteins, which bind and hydrolyze ATP. The HSP90 family of proteinsincludes four known members: Hsp90 α and β, Grp94 and Trap-1. The rolesof HSP90s in cellular functions are not completely understood, butrecent studies indicate that HSP90s are involved in folding, activationand assembly of a wide range of proteins, including key proteinsinvolved in signal transduction, cell cycle control and transcriptionalregulation. For example, researchers have reported that HSP90 chaperoneproteins are associated with important signaling proteins, such assteroid hormone receptors and protein kinases, including many implicatedin tumorigenesis, such as Raf-1, EGFR, v-Src family kinases, Cdk4, andErbB-2 (Buchner J., 1999, TIBS, 24:136-141; Stepanova, L. et al., 1996,Genes Dev. 10:1491-502; Dai, K. et al., 1996, J. Biol. Chem.271:22030-4).

In vivo and in vitro studies indicate that without the aid ofco-chaperones HSP90 is unable to fold or activate proteins. For steroidreceptor conformation and association in vitro, HSP90 requires Hsp70 andp60/Hop/Sti1 (Caplan, A., 1999, Trends in Cell Biol., 9: 262-68). Invivo HSP90 may interact with HSP70 and its co-chaperones. Otherco-chaperones associated with HSP90s in higher eukaryotes include Hip,Bag1, HSP40/Hdj2/Hsj1, Immunophillinis, p23, and p50 (Caplan, A. supra).

Ansamycin antibiotics are natural products derived from Streptomyceshygroscopicus that have profound effects on eukaryotic cells. Manyansamycins, such as herbimycin A (HA) and geldanamycin (GM), bindtightly to a pocket in the HSP90 (Stebbins, C. et al., 1997, Cell,89:239-250). The binding of ansamycins to HSP90 has been reported toinhibit protein refolding and to cause the proteasome dependentdegradation of a select group of cellular proteins (Sepp-Lorenzino, L.,et al., 1995, J. Biol. Chem., 270:16580-16587; Whitesell, L. et al.,1994, Proc. Natl. Acad. Sci. USA, 91: 8324-8328).

The ansamycins were originally isolated on the basis of their ability torevert v-src transformed fibroblasts (Uehara, Y. et al., 1985, J. CancerRes., 76: 672-675). Subsequently, they were said to haveantiproliferative effects on cells transformed with a number ofoncogenes, particularly those encoding tyrosine kinases (Uehara, Y., etal., 1988, Virology, 164: 294-98). Inhibition of cell growth isassociated with apoptosis and, in certain cellular systems, withinduction of differentiation (Vasilevskaya, A. et al., 1999, CancerRes., 59: 3935-40). A GM derivative is currently in phase I clinicaltrials.

The use of ansamycins as anticancer agents are described in U.S. Pat.Nos. 4,261,989, 5,387,584 and 5,932,566. The preparation of theansamycin, geldanamiycin, is described in U.S. Pat. No. 3,595,955(incorporated herein by reference).

The ansamycin-binding pocket in the N-terminus of Hsp90 is highlyconserved and has weak homology to the ATP-binding site of DNA gyrase(Stebbins, C. et al., supra; Grenert, J. P. et al., 1997, J. Biol.Chem., 272:23843-50). This pocket has been reported to bind ATP and ADPwith low affinity and to have weak ATPase activity (Proromou, C. et al.,1997, Cell, 90: 65-75; Panaretou, B. et al., 1998, EMBO J., 17:4829-36). In vitro and in vivo studies are said to indicate thatoccupancy of the pocket by ansamycins alters HSP90 function and inhibitsprotein refolding. At high concentrations, ansamycins have been reportedto prevent binding of protein substrates to HSP90 (Scheibel, T., H. etal., 1999, Proc. Natl. Acad. Sci. USA 96:1297-302; Schulte, T. W. etal., 1995, J. Biol. Chem. 270:24585-8; Whitesell, L., et al., 1994,Proc. Natl. Acad. Sci. USA 91:8324-8328). Alternatively, they have alsobeen reported to inhibit the ATP-dependent release ofchaperone-associated protein substrates (Schleider, C., L. et al., 1996,Proc. Natl. Acad. Sci. USA, 93:14536-41; Sepp-Lorenzino et al., 1995, J.Biol. Chem. 270:16580-16587). In both models, the unfolded substratesare said to be degraded by a ubiquitin-dependent process in theproteasome (Schneider, C., L., supra; Sepp-Lorenzino, supra.)

In both tumor and nontransformed cells, binding of ansamycins to HSP90has been reported to result in the degradation of a subset of signalingregulators. These include Raf (Schulte, T. W. et al., 1997, Biochem.Biophys. Res. Commun. 239:655-9; Schulte, T. W., et al., 1995, J. Biol.Chem. 270:24585-8), nuclear steroid receptors (Segnitz, B., and U.Gehring. 1997, J. Biol. Chem. 272:18694-18701; Smith, D. F. et al.,1995, Mol. Cell. Biol. 15:6804-12), v-src (Whitesell, L., et al., 1994,Proc. Natl. Acad. Sci. USA 91:8324-8328) and certain transmembranetyrosine kinases (Sepp-Lorenzino, L. et al., 1995, J. Biol. Chem.270:16580-16587) such as EGF receptor (EGFR) and Her2/Neu (Hartmann, F.,et al., 1997, Int. J. Cancer 70:221-9; Miller, P. et al., 1994, CancerRes. 54:2724-2730; Mimnaugh, E. G., et al., 1996, J. Biol. Chem.271:22796-801; Schnur, R. et al., 1995, J. Med. Chem. 38:3806-3812). Theansamycin-induced loss of these proteins is said to lead to theselective disruption of certain regulatory pathways and results ingrowth arrest at specific phases of the cell cycle (Muise-Heimericks, R.C. et al., 1998, J. Biol. Chem. 273:29864-72).

Cyclin D in complex with Cdk4 or Cdk6 and cyclin E-Cdk2 phosphorylatethe protein product of the retinoblatoma gene, Rb. Researchers havereported that the protein product of the Rb gene is a nuclearphosphoprotein, which arrests cells during the G₁ phase of the cellcycle by repressing transcription of genes involved in the G₁ to S phasetransition (Weinberg, R. A., 1995, Cell, 81:323-330). DephosphorylatedRb is said to inhibit progression through late G₁, in part, through itsinteraction with E2F transcription family members, which ultimatelyrepresses the transcription of E2F target genes (Dyson, N., 1998, GenesDev., 12: 2245-2262). Progressive phosphorylation of Rb by thecyclin-dependent kinases in mid to late G₁ leads to dissociation of Rbfrom Rb-E2F complexes, allowing the expression of E2F target genes andentry into the S phase.

The retinoblastoma gene product is mutated in several tumor types, suchas retinoblastoma, osteosarcoma and small-cell lung cancer. Researchalso indicates that in many additional human cancers the function of Rbis is disrupted through neutralization by a binding protein, (e.g., thehuman papilloma virus-E7 protein in cervical carcinoma; Ishiji, T, 2000,J Dermatol., 27: 73-86) or deregulation of pathways ultimatelyresponsible for its phoshorylation. Inactivation of the Rb pathway oftenresults from pertubation of p16INK04a, Cyclin D1, and Cdk4.

The retinoblastoma gene product, besides being a target of humanpapilloma E7 protein, is also the target of other oncogenic viral geneproducts. For example, studies indicate that the simian virus 40 large Tantigen inactivates the Rb family of proteins, including Rb, p107, andp130 (Zalvide, J. H. et al., 1998, Mol. Cell. Biol., 18: 1408-1415).Research also indicates that transformation by adenovirus requires E1Abinding to Rb (Egan, C. et al., 1989, Oncogene, 4:383-388).

Scientists estimate that over 70 types of papilloma viruses infecthumans (HPV) (Sasagawa, T. et al., 1996, Clinical Diag. Lab. Immunol, 3:403-410). Of these several are associated with malignancies of humans,particularly cervical cancers (Bosch et al., 1995, J. Natl. CancerInst., 87:796-802). Recent evidence also implicates HPV in some head andneck cancers. Several types of HPV are associated with an intermediateto high risk of malignancies (types 16, 18, 31, 33, 35, 45, and 56)(Sasagawa, T., et al., supra). In infections with these HPV, the viralgenome integrates into the genome of the infected cell with subsequentexpression of transforming genes E6 and E7. Data indicate that theproducts of these genes may promote malignant transformation by alteringthe functions of two cellular tumor suppressor proteins (p53 and Rb). E6causes the proteolytic degradation of p53 (Scheffiner, M. et al., 1990,Cell, 63: 1129-1136. E7 complexes with Rb causing its release fromtranscription factor E2F, leading to the activation of genes involved incell proliferation (Dyson, N. et al., 1988, Science, 243: 934-937.).

Most cancer therapies are not successful with all types of cancers. Forexample, solid tumor types ultimately fail to respond to eitherradiation or chemotherapy. There remains a need for cancer treatmentswhich target specific cancer types. The present invention satisfiesthese needs and provides related advantages as well. The presentinvention provides novel methods for treating cell proliferativedisorders and viral infections associated with retinoblastoma negativeor deficient cells.

SUMMARY OF THE INVENTION

The present invention relates to methods useful for the treatment of ananimal, preferably a mammal, that has a cell proliferative disorder orviral infection associated with Rb negative or deficient cells. One suchmethod comprises administering an effective amount of a pharmaceuticalcomposition that comprises a pharmaceutically acceptable carrier and acompound that binds to the N-terminal pocket of heat shock protein 90 tocells that are Rb negative or Rb deficient. In a preferred embodimentthe HSP90 binding compound is an ansamycin. In a particularly preferredembodiment, the ansamycin is 17-allylamino-(17)-demethoxygeldanamycin(17-AAG).

The present invention further provides methods of destroying cells thatare deficient in the retinoblastoma gene product. In one suchembodiment, the method comprises administering an effective amount of acompound that binds to the N-terminal pocket of HSP90 to cells that areRb negative or Rb deficient. In one embodiment, the HSP90 bindingcompound is an ansamycin. In a particularly preferred embodiment, theansamycin is 17-AAG.

In another embodiment, the invention provides a method of destroying Rbnegative or Rb deficient cells, comprising administering an effectiveamount of a compound that binds to the N-terminal pocket of HSP90selected from the group consisting of herbimycin, geldanamycin, and17-AAG, radicicol or synthetic compounds that bind into the N-terminalpocket of HSP90 which is the ATP-binding site of HSP90.

The method can further comprise treating a mammal in combination withother therapies. Other such therapies include, but are not limited to,chemotherapy, surgery, and/or radiotherapy.

By means of the invention, a method of destroying cells which are Rbnegative or Rb deficient is provided. The invention provides a means totreat cell proliferative disorders, tumors associated with viralinfections and certain viral infections associated with an Rb negativephenotype. These and other advantages of the present invention will beappreciated from the detailed description and examples set forth below.The detailed description and examples enhance the understanding of theinvention, but are not intended to limit the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows differential cell cycle effects of Herbimycin on Rb-wildtype (A) and Rb-negative cells (B). (A) MCF7 and Colo 205; (B) MB-MDA468 and BT 549

FIG. 2 shows levels of mitotic cyclin expression and associated kinaseactivities in Herbimycin arrested MB-MD 468 cells. FIG. 2(A) shows awestern blot using anti-cyclin A and also shows an in vitro kinase assayof immunoprecipitates isolated with anti-cyclin A. FIG. 2(B) shows awestern blot using anti-cyclin B1 antibodies and also shows an iii vitrokinase assay of immunoprecipitates isolated with anti-cyclin B1.

FIG. 3 shows Rb-wild-type cells complete mitosis in the presence of HAafter arrest with aphidicolin (FIG. 3A). FIG. 3B shows that, afterrelease from aphidicolin, Rb-negative MB-MDA 468 cells arrested in thenext mitosis

FIG. 4A and B shows that HA induces mitotic arrest and not G₁ arrest inprimary cells expressing HPV 16 E6 and E7.

FIG. 5 shows the effect of HA on Rb-negative cells transfected with theRb gene. FIG. 5A shows a western blot analysis of Rb expression inMB-MDA 468, 468-7 and 468-19 FIGS. 5B-D show that introduction of the Rbgene abrogates HA-induced mitotic arrest in MB-MDA 468 cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns the surprising discovery that ansamycinscause Rb negative or Rb deficient cells to undergo mitotic arrestfollowed by rapid programmed cell death. This is in contrast toansamycin treatment of cells containing wild-type levels of Rb, whichcauses cells to arrest in G₁ of the cell cycle followed, in some cases,by differentiation and apoptosis. The induction of mitotic arrest byansamycins in Rb negative or Rb deficient cells, which rapidly leads toprogrammed cell death, is a phenomenon confined to cells with defectiveRb function. Mitosis is unaffected in normal cells with wild-type Rb.Thus, the present invention will aid in the treatment of cellproliferative disorders which are associated with Rb negative or Rbdeficient cells, such as small-cell lung cancers, retinoblastoma,osteosarcoma, certain breast cancers, prostate cancer, bladder cancer,hepatocarcinoma, certain viral infections, and virally induced tumors,including those caused by human papilloma viruses, such as cervicalcarcinoma.

As used in the specification and claims of this application, the term“Rb deficient” describes several types of cells, including cells whichproduce no detectable amounts of a functional Rb protein. Such cells arereferred to herein as “Rb negative” cells. Cells which are Rb deficientmay be cells which do not contain a functional Rb gene. Cells which areRb deficient may also be cells that can encode an Rb protein, but inwhich the protein does not function properly or is produced at lowerthan normal level. An Rb deficient phenotype can also occur due to theperturbation of the pathway which ultimately results in phosphorylationof the Rb protein, for example, perturbation of p16INK4a, Cyclin D1, orCdk4, and cells with such a perturbation are Rb deficient cells.

As used in the specification and claims of this application, the term“HSP90” refers to the family of HSP90 heat shock proteins. Thus, thisterm encompasses Hsp90 α and Hsp90β, Grp94 and Trap-1. The HSP90 heatshock proteins each possess a characteristic pocket located near theN-terminal end of the protein to which ATP and ADP bind. This is thesame pocket which has been shown to bind to ansamycin antibiotics. Thispocket is referred to herein as “the N-terminal pocket of HSP90”.

Although the precise mechanisms are not yet understood, the presentapplication makes use of compositions that bind to the N-terminal pocketof HSP90 in a manner that results in an alteration of the function ofHSP90. As used in the specification and claims of this application, thisalteration of function is referred to as “inhibition of HSP90 function”.In accordance with the present invention, this inhibition occurs uponadministration of HSP90 binding compounds, such as ansamycins, andresults in arrest of Rb negative or deficient cells in mitosis. Suchcells uniformly die through apoptotic mechanisms. This novel mechanismof destroying cells that are Rb negative or deficient provides a meansto specifically treat cell proliferative disorders and certain viralinfections associated with cells that are Rb negative or deficient.

The destruction of Rb negative or deficient cells can occur with lesscytotoxicity to normal cells or tissues. For example, when cells whichcontain a normal Rb gene product are treated with HSP90 inhibitors,those cells arrest in G₁ of the cell cycle and, in some cases, maydifferentiate and die. However, cells which are Rb negative or deficientuniformly die when treated with HSP90 inhibitors. Further, such cellswill be more susceptible to other agents or radiation treatments andwill require lower doses of drug for killing than cells with wild-typeretinoblastoma gene product. Studies indicate that the G₂/M phase of thecell cycle is the most radiosensitive phase of the cell cycle (Sinclair,W. K, 1968, Radiat. Res., 33:620).

In one embodiment of the invention, the IC₅₀ of the HSP90 inhibitor usedin the instant methods to destroy cells which are Rb negative of Rbdeficient is lower than the IC₅₀ against similar cells which are not Rbnegative or deficient. Preferably the IC₅₀ is 5-fold lower, morepreferably 10-fold lower, still further 20-fold lower, and mostpreferably 30- to 50-fold lower when compared to similar cellscontaining wild-type Rb.

As used herein “IC₅₀” is defined as the concentration of an HSP90inhibitor required to achieve killing of 50% of cells.

The term “effective amount” as used herein, means an amount of acompound utilized in the methods of the present invention which iscapable of providing a therapeutic effect. The specific dose of compoundadministered according to this invention to obtain therapeutic and/orprophylactic effects will, of course, be determined by the particularcircumstances surrounding the case, including, for example, the compoundadministered, the route of administration, the condition being treatedand the individual being treated. A typical daily dose (administered insingle or divided doses) will contain a dosage level of from about 0.01mg/kg to about 50 mg/kg of body weight of an active compound of thisinvention. Preferred daily doses generally will be from about 0.05 mg/kgto about 20 mg/kg and ideally from about 0.1 mg/kg to about 10 mg/kg.

The preferred therapeutic effect of the methods of the instantinvention, with respect to cell proliferative disorders is theinhibition, to some extent, of growth of cells causing or contributingto a cell proliferative disorder. A therapeutic effect relieves to someextent one or more of the symptoms of a cell proliferative disorder. Inreference to the treatment of a cancer, a therapeutic effect refers toone or more of the following: 1) reduction in the number of cancercells; 2) reduction in tumor size; 3) inhibition (i.e., slowing to someextent, preferably stopping) of cancer cell infiltration into peripheralorgans; 3) inhibition (i.e., slowing to some extent, preferablystopping) of tumor metastasis; 4) inhibition, to some extent, of tumorgrowth; and/or 5) relieving to some extent one or more of the symptomsassociated with the disorder.

In reference to the treatment of a cell proliferative disorder otherthan a cancer, a therapeutic effect refers to either: 1) the inhibition,to some extent, of the growth of cells causing the disorder; 2) theinhibition, to some extent, of the production of factors (e.g., growthfactors) causing the disorder; and/or 3) relieving to some extent one ormore of the symptoms associated with the disorder.

With respect to viral infections, the preferred therapeutic effect isthe inhibition of a viral infection. More preferably, the therapeuticeffect is the destruction of cells which contain the virus.

The methods of this invention are useful for inhibiting cellproliferative diseases associated with Rb negative or Rb deficient, forexample, retinoblastoma, osteosarcoma, breast cancers, bladder cancer,prostate cancer, renal carcinoma, cancers associated with viralinfections, such as cervical cancers associated with human papillomavirus, and small-cell lung cancer. Additionally, the methods of theinvention are useful for the treatment of certain viral infections whichresult in an Rb negative phenotype, such as human papilloma virus.

“Cell proliferative disorders” refer to disorders wherein unwanted cellproliferation of one or more subset(s) of cells in a multicellularorganism occurs, resulting in harm, for example, pain or decreased lifeexpectancy to the organism. Cell proliferative disorders include, butare not limited to, tumors, benign tumors, blood vessel proliferativedisorders, autoimmune disorders and fibrotic disorders.

The methods of the present invention may be used on mammals, preferablyhumans, either alone or in combination with other therapies or methodsuseful for treating a particular cell proliferative disorder or viralinfection.

The use of the present invention is facilitated by first identifyingwhether the cell proliferation disorder or viral infection isaccompanied by cells which contain altered expression of the Rb geneproduct. Once such disorders are identified, patients suffering fromsuch a disorder can be identified by analysis of their symptoms byprocedures well known to medical doctors. Such patients can then betreated as described herein.

The determination of whether the cell proliferation disorder isassociated with an altered expression of the Rb gene product call becarried out by first determining the protein expression of Rb in theappropriate cells isolated from a mammal suspected of having a cellproliferative disorder or viral infection. For example, in the case ofsmall-cell lung cancer, the protein expression of Rb determined fromcells isolated from a mammal suspected of having small cell lung cancercan be compared to the appropriate cells isolated from a disease freemammal. Rb expression and/or mutations can be measured using methodswell known in the art, including, but not limited to,immunohistochemistry, Southern blot analysis, and Northern blotanalysis. The use of immunohistochemistry (e.g., Western blot analysis)to determine Rb expression is described by Higashiyam M et al., 1994,Oncogene, 51: 544-51, and Kohn G. J et al., 1997, J. Gasroeterol.Hepatol., 12: 198-203, both of these references are incorporated hereinby reference in their entireties. The use of Southern blot analysis todetermine defects in the Rb gene is demonstrated by Presti J. C. Jr. etal., 1996, Anticancer Res., 16:549-56, which is incorporated herein byreference in its entirety. The determination of Rb mRNA using Northernblot analysis is demonstrated by Rygaard K. et al., 1990, Cancer Res.,50: 5312-7, which is incorporated by reference herein in its entirety.If the analysis indicates that there is altered Rb expression, thepatient is a candidate for treatment using the methods described herein.

In the case of cell proliferative disorders arising due to unwantedproliferation of non-cancer cells, the level of the Rb gene product iscompared to that level occurring in the general population (e.g., theaverage level occurring in the general population of people or animalsexcluding those people or animals suffering from a cell proliferativedisorder). If the unwanted cell proliferation disorder is characterizedby an abnormal level of Rb than occurring in the general population,then the disorder is a candidate for treatment using the methodsdescribed herein.

Methods to determine HPV association of with cervical cancer aredescribed in Sasagawa, T. et al., supra, which is incorporated herein byreference.

Cell proliferative disorders, including those referenced above are notnecessarily independent. For example, fibrotic disorders may be relatedto, or overlap with, blood vessel disorders. Additionally, for example,atherosclerosis (which is characterized herein as a blood vesseldisorder) is associated with the abnormal formation of fibrous tissue.

A cancer cell refers to various types of malignant neoplasms, most ofwhich can invade surrounding tissues, and may metastasize to differentsites, as defined by Stedman's Medical Dictionary 25th edition (Hensyled. 1990).

The formation and spreading of blood vessels, or vasculogenesis andangiogenesis respectively, play important roles in a variety ofphysiological processes such as embryonic development, wound healing andorgan regeneration. They also play a role in cancer development. Bloodvessel proliferation disorders refer to angiogenic and vasculogenicdisorders generally resulting in abnormal proliferation of bloodvessels. Examples of such disorders include restenosis, retinopathies,and atherosclerosis.

As noted above, other such proliferative diseases can be identified bystandard techniques, and by determination of the efficacy of action ofthe compounds described herein.

A. Rb Negative or Deficient Cells Arrest in Mitosis After Treatment WithAnsamycins

Rb negative or deficient cells treated with ansamycin or radicicol werediscovered to contain a bipolar spindle and elevated cyclinB1-associated kinase activity. However, chromosomal alignment wasdisorganized, with chromosomes scattered along the length of thespindle. The presence of paired chromosomes at the poles led to theconclusion that HA-treated cells had arrested in prometaphase as aresult of failure of chromosomes to align into a metaphase plate. Thisarrest was dependent on the absence of Rb as introduction of wild-typeRB allowed progression through mitosis in the presence of drug. Whentreated with ansamycins in S phase, Rb-negative cells blocked in thesubsequent mitosis whereas Rb-wild type cells progressed through mitosisand arrested in G. Thus, Rb is required for completion of mitosis whenHsp90 function is inhibited.

In 12 tumor cell lines examined, ansamycin treatment caused growtharrest in G₁ (FIG. 1A). This arrest was accompanied by a rapid declinein D-cyclin-associated kinase activity and hypophosphorylation of Rb,suggesting that ansamycins affect G₁ via a cyclin D-related pathway(Srethapakdi, M., F. Liu, R. Tavorath, and N. Rosen, 2000, Cancer Res.60: 3940-6). These effects were elicited by three different ansarnycins,HA, GM and its derivative, 17-allylamino-(17)demethoxygeldanamycin(17-AAG), differing only in regard to potency. Although theseexperiments were done, for the most part, with HA, it will be understoodthat similar effects can be obtained using other ansamycins, which bindto the HSP90 pocket, such as the benzoquinone ansamycins, including, butnot limited to, geldanamycin, geldanamycin derivatives, such as 17-AAG,herbimycin, and macbecins, or other compounds which bind to the HSP 90pocket, such as radicicol. To determine if ansamycins disrupted G₁,progression by inhibiting the cyclin D-Rb pathway, their effects wereexamined in cell lines lacking functional Rb. Rb is the only knownsubstrate of cyclin D-associated kinases (Baldin, V., et al., 1993,Genes Dev. 7:812-21; Ewen, M. E. et al., 1993, Cell 73:487-97; Kato, J.,H. et al., 1993, Genes Dev. 7:331-42; Matsushime, H., et al., 1992, Cell71:323-34; Matsushime, H., D. et al., 1994, Mol. Cell. Biol. 14:2066-76;Meyerson, M., and E. Harlow, 1994, Mol. Cell. Biol. 14:2077-86; Quelle,D. E., et al., 1993, Genes Dev. 7:1559-1571; Guan, K. -L., et al., 1994,Genes Dev. 8:2939-52; Koh, J. et al., 1995, Nature 375:506-10; Lukas, J.et al., 1995, Mol. Cell. Biol. 15:2600-1 1; Lukas, J., H. et al., 1994,J. Cell Biol. 125:625-38; Lukas. J., D. et al., 1995, Nature 375:503-6;Medema, R. H. et al., 1995, Proc. Natl. Acad. Sci. USA, 92:6289-93). Intumor cell lines with mutated Rb (MB-MDA 468, BT-549, DU145 and DU4475)HA treatment failed to induce a G₁ block but instead led to anaccumulation of cells with 4n DNA content (FIG. 1B).

To determine if HA treatment caused Rb-negative cells to arrest in G₂ ormitosis, mitotic index was determined with bisbenzimide staining andmitosis was scored by the presence of condensed chromosomes. In MB-MDA468 cells, in which the mitotic index of the control population was5-10%, 60-70% of HA-treated cells were in mitosis. Thus, in the absenceof Rb function, HA treatment resulted in mitotic arrest.

To further define the nature of the HA-induced mitotic defect, cellswere triple-stained with bisbenzimide, anti-α-tubulin antibodies andanti-centromere autoimmune serum (ACA/CREST). α-tubulin stainingrevealed that arrested cells contained bipolar spindles, demonstratingthat HA does not interfere with spindle formation. Examination ofcluomosomnal distribution by bisbenzamide and ACA/CREST staining,however, showed that in most cells, chromosomes localized both to thepoles and within the spindle.

Without being bound to any particular theories, the observedaccumulation of chromosomes at the poles is consistent with either anarrest in prometaphase due to failure of chromosomes to align into ametaphase plate or to an abnormal anaphase with impaired sisterchromatid segregation. ACA staining, however, revealed pairedcentromeres on chromosomes at the poles, indicating that they wereundisjoined sister chromatids (FIG. 3B). In 77 chromosomes localized tothe poles, 87% scored as double dots for ACA staining. This demonstratesthat accumulation of chromosomes at the poles did not result frompremature or incomplete segregation but rather, failure of pairedchromatids to congress to the spindle equator. These data show thatHA-treated cells are arrested in prometaphase and that, in Rb-negativecells, HA induces mitotic arrest by interfering with chromosomalalignment.

To further distinguish between prometaphase and anaphase, the expressionand -associated kinase activities of the mitotic cyclins were assessed.Levels of cyclin A-associated kinase activity begun to decline inprometaphase while cyclin B associated kinase activity remains elevateduntil anaphase (Furuno, N., N. den Eizen, and J. Pines, 1999, J. CellBiol. 147:295-306; Townsley, F. M., and J. V. Ruderman, 1998, TrendsCell Biol. 8:238-244; Zachariae, W., and K. Nasmyth, 1999, Genes Dev.13:2039-58). As the mitotic index in the HA-blocked population is only60-70%, mitotically arrested cells were enriched by using only theloosely adherent population in which the mitotic index was greater than90%. Cyclin B1-associated kinase activity was elevated 5-fold inHA-treated cells when compared to control and was comparable to thatseen in nocodazole-arrested cells (FIG. 2B). In parallel with kinaseactivity, cyclin B1 protein expression was also increased in HA-treatedcells (FIG. 2B). In contrast, cyclin A expression and its associatedkinase activity were slightly lower in both HA and nocodazole-arrestedcells compared to that in control cells (FIG. 2A). Thus, HA inducesarrest at a point before early anaphase and after prophase whenproteolysis of cyclin A but not cyclin B1, has begun. This result showsthat arrest occurs in prometaphase of mitosis.

Cells with Wild-type RB Traverse Mitosis in the Presence of HA

The HA-induced mitotic block was observed, surprisingly, only in cellslacking wild-type Rb. HA likely causes the degradation of mitoticregulators more slowly than it affects the expression of G₁ regulators.The absence of Rb would abrogate the effects on G₁ and expose themitotic phenotype. The addition of HA to Rb-negative cells blocked in Sphase, then, would fail to cause arrest in the mitosis immediatelyfollowing drug addition. To demonstrate this, Colo 205 cells and MB-MDA,468 cells were arrested in G₁/S with aphidicolin and subsequentlyreleased from block in the presence of either HA or DMSO. In thepresence of HA, Rb-wild type Colo 205 cells progressed through G2 andmitosis and were arrested in the next G₁ (FIG. 3A). In contrast,following release from aphidicolin, Rb-negative MB-MDA 468 cellsarrested within 12 hours in the next mitosis (FIG. 3B). Thus, cellscontaining Rb are able to progress normally through mitosis in thepresence of HA while those lacking Rb function are not.

HA Induces M and Not G, Arrest in Primary Cells Expressing HPV 16 E6 andE7

With regard to whether the above observations could result frommutations in other genes that complement with Rb mutations to causetransformation, the cell cycle effects of HA were examined in primaryhuman foreskin keratinocytes (HFK) expressing human papilloma virus-16(HPV-16) E6 and E7. These viral oncogenes functionally inactivate p53and Rb, respectively. Introduction of both E6 and E7 was necessary asloss of Rb function in a p53 wild-type background has been shown topredispose cells to undergo apoptosis (Jones, D. L., D. A. Thompson, andK. Munger, 1997, Virology, 239:97-107; Pan, H., and A. E. Griep, 1994,Genes Dev. 8:1285-99; White, A. E., E. M. Livanos, and T. D. Tlsty,1994, Genes Dev. 8:666-77). While HA caused the majority of primary HFKcells (FIG. 4A) to accumulate in G₁, E6/E7 transfectants arrested with4n DNA content (FIG. 4B). These results provide further evidence thatthe cell cycle response to the HA is dictated by the status of Rb andmoreover, that Rb is required for mitotic traversal following drugexposure. The loss of p53 function alone is not sufficient for mitoticblock as the multiple p53-negative/Rb-positive cell lines that have beentested successfully traverse mitosis in the presence of ansamycins.

Introduction of Rb into Rb-negative Cells Allows Progression Through Min the Presence of HA

The discovery that addition of HA to cells in S phase induces mitoticarrest in Rb negative MB-MDA 468, cells but not Rb-wild type Colo 205cells indicates that Rb permits progression through mitosis under theseconditions. To test this, wild type Rb was reintroduced into the cellline MB-MDA 468. A low transfection efficiency was seen, possiblybecause elevated expression of Rb inhibits cell growth. Five positiveclones were ultimately obtained. These transfectants expressed lowerlevels of Rb when compared to Rb-wild-type tumor cell lines. Two stablytransfected clones expressing different levels of Rb (468-7 and 468-19)were chosen for analysis (FIG. 5A). FACS analysis of logarithmicallygrowing populations revealed that Rb expression in these clones did notalter the cell cycle distribution, though the cells had slightly longerdoubling times. When treated with HA, control transfectants accumulatedwith 4n DNA content. In contrast, the drug had no effect on G₂/M in theRb-transfectants and instead caused an increase in G₁. Furthermore, whenreleased from aphidicolin block into HA, both clone 468-7 and 468-19cycled through mitosis and entered G₁, (FIGS. 5C & D). In contrast, whentreated with HA after aphidicolin block, MB-MDA 468 cells failed toreach G₁ and arrested in mitosis by 12 hours (FIG. 5D). The amount ofcell death induced by ansamycins was comparable in the Rb-transfectedand untransfected cells. Thus, in the Rb-transfectants, the appearanceof a higher percentage of cells in G₁ does not result from increasedapoptosis of cells in G₂/M. As these cell lines differ only in Rbstatus, this finding demonstrates that Rb expression alone is sufficientto allow progression through mitosis in the presence of HA.

Inhibition of Hsp90 with Radicicol Induces Mitotic Arrest in MB-MDA 468Cells

HA binds to Hsp90 but may have other effects that relate to its chemicalproperties. Treatment with GM and 17-AAG generated the same Rb-dependentcell cycle profiles and mitotic phenotype as observed with HA. Radicicolis a non-ansamycin natural product that has been shown to bind to theN-terminal Hsp90 pocket (Schulte, T. et al., 1999, Mol. Enidocrinol.13:1435-1448; Schulte, T. et al., 1998, Cell Stress Chaperones 3:100-8)and to induce the degradation of the same spectrum of proteins affectedby ansamycins (Soga, S., et al., 1998, J. Biol. Chest. 273:822-828).Radicicol treatment induced G₁ arrest in Rb-positive cell lines, Colo2O5and MCF7, but failed to arrest Rb-negative MDA-468 cells in G₂, andinstead, like ansamycins, caused an accumulation of cells with 4n DNAcontent. Radicicol-arrested MDA 468 cells also displayed chromosomeslocalized to the poles as well as strewn along the spindle.

B. Administration and Pharmaceutical Compositions

The compounds utilized in the methods of the instant invention may beadministered either alone or in combination with pharmaceuticallyacceptable carriers, excipients or diluents, in a pharmaceuticalcomposition, according to standard pharmaceutical practice. Thecompounds can be administered orally or parenterally, including theintraventous, intramuscular, intraperitoneal, subcutaneous, rectal andtopical routes of administration.

The pharmaceutical compositions used in the methods of the instantinvention can contain the active ingredient in a form suitable for oraluse, for example, as tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsions, hard or softcapsules, or syrups or elixirs. Compositions intended for oral use maybe prepared according to any method known to the art for the manufactureof pharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations. Tabletscontain the active ingredient in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,such as microcrystalline cellulose, sodium crosscarmellose, corn starch,or alginic acid; binding agents, for example starch, gelatin,polyvinyl-pyrrolidone or acacia, and lubricating agents, for example,magnesium stearate, stearic acid or talc. The tablets may be uncoated orthey may be coated by known techniques to mask the unpleasant taste ofthe drug or delay disintegration and absorption in the gastrointestinaltract and thereby provide a sustained action over a longer period. Forexample, a water soluble taste masking material such ashydroxypropylmethyl-cellulose or hydroxypropylcellulose, or a time delaymaterial such as ethyl cellulose, cellulose acetate butyrate may beemployed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with watersoluble carrier such as polyethyleneglycol or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene-oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of anantioxidant such as butylated hydroxyanisol or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present. These compositions may be preserved by theaddition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions used in the methods of the instantinvention may also be in the form of an oil-in-water emulsions. The oilyphase may be a vegetable oil, for example olive oil or arachis oil, or amineral oil, for example liquid paraffin or mixtures of these. Suitableemulsifying agents may be naturally-occurring phosphatides, for examplesoy bean lecithin, and esters or partial esters derived from fatty acidsand hexitol anhydrides, for example sorbitan monooleate, andcondensation products of the said partial esters with ethylene oxide,for example polyoxyethylene sorbitan monooleate. The emulsions may alsocontain sweetening, flavoring agents, preservatives and antioxidants.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative, flavoring and coloring agentsand antioxidant.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous solutions. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution and isotonic sodiumchloride solution.

The sterile injectable preparation may also be a sterile injectableoil-in-water microemulsion where the active ingredient is dissolved inthe oily phase. For example, the active ingredient may be firstdissolved in a mixture of soybean oil and lecithin. The oil solutionthen introduced into a water and glycerol mixture and processed to forma microemulation.

The injectable solutions or microemulsions may be introduced into apatient's blood-stream by local bolus injection. Alternatively, it maybe advantageous to administer the solution or microemulsion in such away as to maintain a constant circulating concentration of the instantcompound. In order to maintain such a constant concentration, acontinuous intravenous delivery device may be utilized. An example ofsuch a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension for intramuscular andsubcutaneous administration. This suspension may be formulated accordingto the known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables.

The HSP90 inhibitors used in the methods of the present invention mayalso be administered in the form of a suppositories for rectaladministration of the drug. These compositions can be prepared by mixingthe inhibitors with a suitable nonirritating excipient which is solid atordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Such materials includecocoa butter, glycerinated gelatin, hydrogenated vegetable oils,mixtures of polyethylene glycols of various molecular weights and fattyacid esters of polyethylene glycol.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing an HSP90 inhibitor can be used. (As used herein,topical application can include mouth washes and gargles.)

The compounds used in the methods of the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles and delivery devices, or via transdermal routes, using thoseforms of transdermal skin patches well known to those of ordinary skillin the art. To be administered in the form of a transdermal deliverysystem, the dosage administration will, of course, be continuous ratherthan intermittent throughout the dosage regimen.

The HSP90 inhibitors used in the instant invention may also beco-administered with other well known therapeutic agents that areselected for their particular usefulness against the condition that isbeing treated. For example, the instant compounds may be useful incombination with known anti-cancer and cytotoxic agents. Similarly, theinstant compounds may be useful in combination with agents that areeffective in the treatment and prevention of certain viral infections orother conditions associated with an Rb negative phenotype. The instantcompounds may also be useful in combination with other inhibitors ofparts of the signaling pathway that links cell surface growth factorreceptors to nuclear signals initiating cellular proliferation.

The methods of the present invention may also be useful with otheragents that inhibit angiogenesis and thereby inhibit the growth andinvasiveness of tumor cells, including, but not limited to VEGF receptorinhibitors, including ribozymes and antisense targeted to VEGFreceptors, angiostatin and endostatin.

Examples of an antineoplastic agents, which can be used in combinationwith the methods of the present invention include, in general,alkylating agents, anti-metabolites; epidophyllotoxin; an antineoplasticenzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinumcoordination complexes; biological response modifiers and growthinhibitors; hormonal/anti-hormonal therapeutic agents and haematopoieticgrowth factors.

Example classes of antineoplastic agents includes for example, theanthracycline family of drugs, the vinca drugs, the mitomycins, thebleomycins, the cytotoxic nucleosides, the epothilones, discodermolide,the pteridine family of drugs, diynenes and the podophyllotoxins.Particularly useful members of those classes include, for example,carminomycin, daunorubicin, aminopterin, methotrexate, methopterin,dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil,6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin orpodophyllotoxin derivatives such as etoposide, etoposide phosphate orteniposide, melphalan, vinblastine, vincristine, leurosidine, vindesine,leurosine, paclitaxel and the like. Other useful antineoplastic agentsinclude estramustine, carboplatin, cyclophosphamide, bleomycin,gemcitibine, ifosamide, melphalan, hexamethyl melamine, thiotepa,cytarabin, idatrexate, trimetrexate, dacarbazine, L-asparaginase,camptothecin, CPT-11, topotecan, ara-C, bicalutamide, flutamide,leuprolide, pyridobenzoindole derivatives, interferons and interleukins.

When a HSP90 inhibitor used in the methods of the present invention isadministered into a human subject, the daily dosage will normally bedetermined by the prescribing physician with the dosage generallyvarying according to the age, weight, and response of the individualpatient, as well as the severity of the patient's symptoms.

In one exemplary application, a suitable amount of a HSP90 inhibitor isadministered to a mammal undergoing treatment for cancer. Administrationoccurs in an amount of each type of inhibitor of between about 0.1 mg/kgof body weight to about 60 mg/kg of body weight per day, preferably ofbetween 0.5 mg/kg of body weight to about 40 mg/kg of body weight perday. A particular therapeutic dosage that comprises the instantcomposition includes from about 0.01 mg to about 1000 mg of a HSP90inhibitor. Preferably, the dosage comprises from about 1 mg to about1000 mg of a HSP90 inhibitor.

Preferably, the pharmaceutical preparation is in unit dosage form. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component, e.g., an effectiveamount to achieve the desired purpose.

The quantity of active compound in a unit dose of preparation may bevaried or adjusted from about 0.1 mg to 1000 mg, preferably from about 1mg to 300 mg, more preferably 10 mg to 200 mg, according to theparticular application.

The actual dosage employed may be varied depending upon the requirementsof the patient and the severity of the condition being treated.Determination of the proper dosage for a particular situation is withinthe skill of the art. Generally, treatment is initiated with smallerdosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small amounts until the optimumeffect under the circumstances is reached. For convenience, the totaldaily dosage may be divided and administered in portions during the dayif desired.

The amount and frequency of administration of the HSP90 inhibitors usedin the methods of the present invention and if applicable otherchemotherapeutic agents and/or radiation therapy will be regulatedaccording to the judgment of the attending clinician (physician)considering such factors as age, condition and size of the patient aswell as severity of the disease being treated.

The chemotherapeutic agent and/or radiation therapy can be administeredaccording to therapeutic protocols well known in the art. It will beapparent to those skilled in the art that the administration of thechemotherapeutic agent and/or radiation therapy can be varied dependingon the disease being treated and the known effects of thechemotherapeutic agent and/or radiation therapy on that disease. Also,in accordance with the knowledge of the skilled clinician, thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents (i.e., antineoplastic agent or radiation) on thepatient, and in view of the observed responses of the disease to theadministered therapeutic agents.

Also, in general, the HSP90 inhibitor and the chemotherapeutic agent donot have to be administered in the same pharmaceutical composition, andmay, because of different physical and chemical characteristics, have tobe administered by different routes. For example, the HSP90 inhibitormay be administered orally to generate and maintain good blood levelsthereof, while the chemotherapeutic agent may be administeredintravenously. The determination of the mode of administration and theadvisability of administration, where possible, in the samepharmaceutical composition, is well within the knowledge of the skilledclinician. The initial administration can be made according toestablished protocols known in the art, and then, based upon theobserved effects, the dosage, modes of administration and times ofadministration can be modified by the skilled clinician.

The particular choice of HSP90 inhibitor, and chemotherapeutic agentand/or radiation will depend upon the diagnosis of the attendingphysicians and their judgment of the condition of the patient and theappropriate treatment protocol.

The HSP90 inhibitor, and chemotherapeutic agent and/or radiation may beadministered concurrently e.g., simultaneously, essentiallysimultaneously or within the same treatment protocol) or sequentially,depending upon the nature of the proliferative disease, the condition ofthe patient, and the actual choice of chemotherapeutic agent and/orradiation to be administered in conjunction (i.e., within a singletreatment protocol) with the HSP90 inhibitor.

If the HSP90 inhibitor, and the chemotherapeutic agent and/or radiationare not administered simultaneously or essentially simultaneously, thenthe initial order of administration of the HSP90 inhibitor, and thechemotherapeutic agent and/or radiation, may not be important. Thus, theHSP90 inhibitor may be administered first followed by the administrationof the chemotherapeutic agent and/or radiation; or the chemotherapeuticagent and/or radiation may be administered first followed by theadministration of the HSP90 inhibitor. This alternate administration maybe repeated during a single treatment protocol. The determination of theorder of administration, and the number of repetitions of administrationof each therapeutic agent during a treatment protocol, is well withinthe knowledge of the skilled physician after evaluation of the diseasebeing treated and the condition of the patient. For example, thechemotherapeutic agent and/or radiation may be administered first,especially if it is a cytotoxic agent, and then the treatment continuedwith the administration of the HSP90 inhibitor followed, wheredetermined advantageous, by the administration of the chemotherapeuticagent and/or radiation, and so on until the treatment protocol iscomplete.

Thus, in accordance with experience and knowledge, the practicingphysician can modify each protocol for the administration of a component(therapeutic agent-i.e., HSP90 inhibitor, chemotherapeutic agent orradiation) of the treatment according to the individual patient's needs,as the treatment proceeds.

The attending clinician, in judging whether treatment is effective atthe dosage administered, will consider the general well-being of thepatient as well as more definite signs such as relief of disease-relatedsymptoms, inhibition of tumor growth, actual shrinkage of the tumor, orinhibition of metastasis. Size of the tumor can be measured by standardmethods such as radiological studies, e.g., CAT or MRI scan, andsuccessive measurements can be used to judge whether or not growth ofthe tumor has been retarded or even reversed. Relief of disease-relatedsymptoms such as pain, and improvement in overall condition can also beused to help judge effectiveness of treatment.

The following examples are not limiting and are merely representative ofvarious aspects and features of the present invention. All referencesreferred to above and below are incorporated herein by reference.

EXAMPLES Example 1 Effect of Ansamycins on Cells with a Functional RbProtein and Cells Lacking a Functional Rb Protein

Cell Culture:

The human breast cancer cell lines MB-MDA 468, MCF7 and BT-549 and thecolon carcinoma cell line, Colo 205, were obtained from ATCC. Breastcell lines were maintained in DME-F12 media and Colo 205 cells in RPMI;both media were supplemented with 5% fetal calf serum (BRL), 2 mMglutamine and 50 u/ml each of penicillin and streptomycin. All cellswere incubated at 37° C. in 5% C0₂.

Cells were treated for 24 hours with 250 ng/ml herbimycin A (Gibco)dissolved in DMSO or 435 nM of radicicol (Sigma).

After treatment the nuclei can Do stained wish ethidium bromide andanalyzed by flow cytometry.

Flow Cytometry:

Nuclei were isolated for flow cytometry assays stained with ethidiumbromide and analyzed using a Becton Dickinson fluorescence-activatedcell sorter. Statistical data was obtained using Multicycle programsoftware.

Results:

As shown in FIG. 1, in 12 tumor cell lines examined, ansamycin treatmentcaused growth arrest in G₁. In tumor cell lines with mutated Rb HAtreatment failed to induce a G₁ block but instead resulted to anaccumulation of cells with 4n DNA content (FIG. 1B).

Example 2 Analysis of Cell Arrest in Rb-Negative and Rb-Positive CellsTreated With HSP90 Inhibitors

Mitotic Index:

For mitotic indices, cells were typsinized, washed with PBS and fixedwith 3% paraformaidehyde in PBS for 20 min. Cells were then stained with3 μg/ml bisbenzimide (Hoechst 33258; Sigma) for 15 min and examinedunder fluorescence microscopy. Mitosis was scored by the presence ofcondensed chromosomes.

Immunofluorescent Analysis:

For immunofluorescent analysis, harvested cells were washed with PBS,fixed with methanol for 20 min at −20° C., washed again and blocked for30 min with 2% BSA in PBS. Cells were then stained with anti-α-tubulin(Sigma) and anti-centromere protein antibodies (ACA/CREST) (gift of Dr.J. D. Rattner) in 2% BSA PBS for 1 hour. Following 3 washes with 0.5%BSA in PBS, cells were incubated with anti-human FITC conjugated,antimouse rhodamine conjugated antibodies (Molecular Probes) and 2 μg/mlbisbenzimide in 2% BSA in PBS for 45 min. Cells were then washed 4× with0.5% BSA in PBS, resuspended in PBS and images captured by confocalmicroscopy or with a CCD camera. Images were then processed usingSlidebook 3.0 and Adobe Photoshop program software.

For synchrony experiments, cells were treated with 1 μg/ml aphidicolin(Sigma) for 18 hours, washed and replated in media containing DMSO orHA.

Results:

As shown in FIG. 3, α-tubulin staining demonstrates that arrested cellscontained bipolar spindles, indicating that ansamycins do not interferewith spindle formation. Additionally, in most cells, chromosomeslocalized both to the poles and within the spindle (FIG. 3A): ACAstaining revealed paired centromeres on chromosomes at the poles (FIG.3B). In 77 chromosomes localized to the poles, 87%, scored as doubledots for ACA staining, indicating that accumulation of chromosomes atthe poles is not the result of premature or incomplete segregation butrather, failure of paired chromatids to assemble to the spindle equator.These data show that HA-treated cells are arrested in promethaphase andthat, in Rb-negative cells, HA induces mitotic arrest by interferingwith chromosomal alignment.

Example 3 Measurement of Mitotic Cyclins

Immunoblot Analysis:

Levels of mitotic cyclin expression and associated kinase activities inherbimycin arrested MB-MD468 cells were assessed using immunoblotanalysis and in vitro kinase assays as described below. Cells culturedwith herbimycin were enriched for mitotically arrested cells by usingonly the loosely adherent population in which the mitotic index wasgreater than 90%.

Immunoblot analysis of lysates from cells treated with DMSO, nocodazoleor herbimycin were analyzed by Western blot analysis using anti-cyclin Aor anti-B1 antibodies. Treated cells were harvested, washed with PBS andlysed in NP40 lysis buffer (50 mM Tris pH7.4, 1% NP40,150 mM NaCl, 40 mMNaF. 1 mM Na₃VO₄, 1 mM phenylmethylsulfonlylfluoride, and 10 μg/ml. eachof leupeptin, aprotinin and soybean trypsin inhibitor) for 30 min onice. Lysates were centrifuged at 15,000×g for 10 min and proteinconcentration determined by bicinchoninic acid protein assay (Pierce).Equal amounts of total protein were resolved by SDS-PAGE and transferredonto Immobilon PVDF membranes (Millipore) by electroblotting. Blots wereblocked overnight in 5% nonfat milk in TBS-T (0.1% Tween-20 TBS, 10 mMTris pH 7.4, 150 mM NaCl) at 4° C. and subsequently probed with eitheranti-cyclin A or cyclin B1 antibodies (Santa Cruz Biotechnology).Following incubation with HRP-conjugated secondary antibodies, proteinswere detected by chemiluminescence (Amersham).

Immunoprecipitation and In Vitro Kinase Assays:

For immunoprecipitation, 100 μg of total protein was incubated withanticyclin A or anti-cyclin B1 (Santa Cruz) antibodies for 2 hours at 4°C. and then for 1 hour following the addition of protein A-Sephaliose.The immune complexes were washed 4× with lysis buffer and boiled inSDS-PAGE sample buffer for 5 min. Following SDS-PAGE, proteins weretransferred onto Immobilon and analyzed by western blotting.

For in vitro kinase reactions, immune complexes were washed 4× withlysis buffer, 2× with kinase buffer (20 mM Tris pH 7.4, 7.5 mM MgCl₂, 1mM DTT) and incubated in 40 μl of kinase buffer containing 2 μg histoneH1, 10 μCi [γ-³²P] ATP and 300 μM ATP for 10 min at 37° C. The reactionwas stopped by the addition of SDS-PAGE sample buffer and boiled for 5min. Proteins were resolved on SDS-PAGE, transferred onto Immobilon andexposed to autoradiography film or phosphoimager screen. Kinase activitywas quantitated by FUJIX phosphoimager and MacBAS program software.

Results:

Cyclin B1-associated kinase activity was elevated 5-fold in HA-treatedcells when compared to control and was comparable to that seen innocodazole-arrested cells (FIG. 4B). Cyclin B1 protein expression wasalso increased in HA-treated cells (FIG. 4B). In contrast, cyclin Aexpression and its associated kinase activity were slightly lower inboth HA and nocodazole-aresseted cells compared to that in control cells(FIG. 4A).

Example 4 Effect of Herbimycin in Cells Expressing Human PapilommaVirus-16 E6 and E7

Primary human foreskin keratinocytes transfected with HPV-1 6 E6 and E7were provided by Drs. H. Stöppler and R. Schlegel (Georgetown Univ.) andgrown as previously described. Primary human foreskin keratinocytes orHPV 16 E6/E7 transfected human foreskin keratinoytes were treated withHA or DMSO for 24 hours and ethidium bromide stained-nuclei analyzed byflow cytometry as described above.

Results:

HA caused the majority of primary HFK cells (FIG. 6A) to accumulate inG₁, in contrast E6/E7 transfectants arrested with 4n DNA content (FIG.6B).

Example 5 Transfection of Rb Gene into Rb-Negative Cells

Rb Transfection:

To further confirm that the gene product of the Rb gene permitsprogression through mitosis in the presence of an HSP90 inhibitor,MB-MDA 468 cells were transfected with the plasmid pUHD1 0-3HGRcontaining full-length 4.7 kb human Rb cDNA. Rb transfectants were grownin DME-F12 media supplemented with 5% fetal calf serum (BRL), 2 mMglutamine and 50 μg/ml each of penicillin and streptomycin and 100 μg/mlhygromycin B (Boehringer Mannheim). To confirm the presence of the Rbgene product Western blot analysis was carried out using anti-Rbantibodies (Pharmingen) as described herein. Vector control, MB-MDA 468,and Rb transfected MB-MDA 468 cells were arrested with aphidicolin asdescribed above. After release from aphidicolin arrest, transfected andnon-transfected MM-MDA 468 cells were cultured in the presence of anansamycin inhibitor as described in Example 1. Cell progression wasmonitored by flow cytometric analysis of ethidium bromide-stained nucleias described above.

Results:

When treated with HA, control transfectants (MB-MDA 468 cells)accumulated with 4n DNA content. In contrast, in the Rb transfectants(468-7; 468-19) HA caused an increase in G₁ and had no effect on G₂/M(data not shown). When released from aphidicolin block into HA, Rbtransfectants cycled through mitosis and entered G₁ (FIGS. 5C-5D). Incontrast, when treated with HA after aphidicolin block, MB-MDA 468 cellsfailed to reach G₁ and arrested in mitosis by 12 hours (FIG. 5B).

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually. None of thereferences are admitted to be prior art.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twotern's. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Other embodiments are within the following claims.

1. A method for destroying cells that are deficient in retinoblastomagene product, comprising administering to said cells a compound capableof inhibiting HSP90 function whereby said cells are destroyed.
 2. Themethod of claim 1, wherein said compound is an ansamycin.
 3. The methodof claim 2, wherein said ansamycin is selected from the group consistingof geldanamycin, 17-AAG, or herbimycin A.
 4. The method of claim 2,wherein said ansamycin is 17-AAG.
 5. The method of claim 1, wherein saidcompound is radicicol.
 6. The method of claim 1, wherein said compoundis a synthetic compound that binds into the ATP-binding site of a HSP90.7. A method of treating disorders associated with cells that aredeficient in retinoblastoma gene product, comprising administering atherapeutically effective amount of a compound capable of inhibitingHSP90, thereby reducing the number of cells that are deficient inretinoblastoma gene product.
 8. A method of treating disordersassociated with cells that are deficient in retinoblastoma gene product,comprising administering a therapeutically effective amount of acompound capable of inhibiting HSP90, thereby reducing the number ofcells that are deficient in retinoblastoma gene product, wherein saiddisorder is small cell lung cancer.
 9. The method of claim 7, whereinsaid disorder is associated with a viral infection.
 10. The method ofclaim 9, wherein said viral infection is caused by a Human papillomavirus.
 11. The method of claim 9, wherein said disorder is cervicalcancer.
 12. The method of claim 7, wherein said compound is given incombination with another therapy for treating the disorder.
 13. Themethod of claim 7, wherein said compound is an ansamycin.
 14. The methodclaim 13, wherein said ansamycin is selected from the group consistingof geldanamycin, 17-AAG, or herbimycin A.
 15. The method of claim 13,wherein said ansamycin is 17-AAG.
 16. The method of claim 7, whereinsaid compound is radicicol.
 17. The method of claim 7, wherein saidcompound is a synthetic compound which binds in the ATP-binding site ofa HSP90.
 18. The method of claim 1, wherein said cells are RB negative.19. The method of claim 7, wherein said cells are RB negative.